A reverse transcriptase is an enzyme that catalyzes RNA dependent DNA synthesis and is an indispensable component of replication-competent retroviruses. Reverse transcriptases direct extension of the 3' end of a DNA strand one deoxynucleotide at a time and cannot initiate synthesis de novo but require a DNA or RNA primer. Typically, reverse transcriptases also possess two additional enzymatic activities: 1) DNA dependent DNA polymerase and 2) RNase H activity. There is a significant amount of research on retroviruses and their inhibitors because of their role in AIDS and other pathologies such as leukemia.
In AIDS research, there are several approaches for antiviral therapy and three classes of antiviral drugs are currently available: nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors. Assays that can detect reverse transcriptase inhibitor activity are important in identifying new inhibitors for use in preventing disease.
Because reverse transcriptase enzymes are essential to replication-competent virions, specifically inhibiting their activity in one method of abating viral propagation. Many methods have been developed for detecting reverse transcriptases, their activity, and their products and the literature can be quite confusing on this. For clarification, a summary of some of the methods is compiled in the table below.
______________________________________ Target Assay Type ______________________________________ Detection of RT protein Antigen capture assay and radioimmunoasssay Detection of RT gene PCR amplification of gene Detection of RT protein Immunoassay Detection of RT enzymatic products PCR and fluorescence detection Detection of RT enzymatic products Direct detection of products Detection of RT enzymatic products RT assay combined with PCR detection of product Detection of RT enzymatic products Microtiter immunoasssay detected modified nucleotides in RT enzymatic product Detection of RT enzymatic products Microtiter assay with immobilized template and primer Detection of RT enzymatic products ELISA and non-isotopic detection method Detection of RT enzymatic products Scintillation Proximity Assay (radioactive) Detection of RT enzymatic products RT assay of unique RNA template combined with PCR detection of product ______________________________________
Gupta, P., Balachandran, R., Grovit, K., Webster, D., and Rinaldo C, J. (1987) Detection of human immunodeficiency virus by reverse transcriptase assay, antigen capture assay, and radioimmunoassay. J Clin Microbiol 25(6), 1122-5; Bruisten, S., van Gemen, B., Koppelman, M., Rasch, M., van Strijp, D., Schukkink, R., Beyer, R., Weigel, H., Lens, P., and Huisman, H. (1993) Detection of HIV-1 distribution in different blood fractions by two nucleic acid amplification assays. AIDS Res Hum Retroviruses 9(3), 259-65; Burger, H., Paul, D., Siegal, F. P., Wendel, I., Neff, S., Eilbott, D., Gehan, K., Grimson, R., and Weiser, B. (1988) Comparison of antigen immunoassay and reverse transcriptase assay for monitoring human immunodeficiency virus infection in an antiviral trial. J Clin Microbiol 26(9), 1890-2; Arnold, B. A., Hepler, R. W., and Keller, P. M. (1998) One-step fluorescent probe product-enhanced reverse transcriptase assay. Biotechniques 25(1), 98-106; Chang, A., Ostrove, J. M., and Bird, R. E. (1997) Development of an improved product enhanced reverse transcriptase assay. J Virol Methods 65(1), 45-54; Ekstrand, D. H., Awad, R. J., Kallander, C. F., and Gronowitz, J. S. (1996) A sensitive assay for the quantification of reverse transcriptase activity based on the use of carrier-bound template and non-radioactive-product detection, with special reference to human-immunodeficiency-virus isolation. Biotechnol Appl Biochem 23(Pt 2)), 95-105; Nakano, T., Sano, K., Odawara, F., Saitoh, Y., Otake, T., Nakamura, T., Hayashi, K., Misaki, H., and Nakai, M. (1994) An improved non-radioisotopic reverse transcriptase assay and its evaluation. Kansenshogaku Zasshi 68(7), 923-3 1; Sano, K., Odawara, F., and Nakai, M. (1996) Comparison of the sensitivities of two non-isotopic reverse transcriptase (RT) assays for human immunodeficiency virus type 1 RT. J Virol Methods 58(1-2), 199-204; Taylor, P. B., Culp, J. S., Debouck, C., Johnson, R. K., Patil, A. D., Woolf, D. J., Brooks, I., and Hertzberg, R. P. (1994) Kinetic and mutational analysis of human immunodeficiency virus type 1 reverse transcriptase inhibition by inophyllums, a novel class of non-nucleoside inhibitors. J Biol Chem 269(9), 6325-31; Yamamoto, S., Folks, T. M., and Heneine, W. (1996) Highly sensitive qualitative and quantitative detection of reverse transcriptase activity: optimization, validation, and comparative analysis with other detection systems. J Virol Methods 61(1-2), 135-43.
Many different types of assays exist for reverse transcriptases. Some of these assays detect the reverse transcriptase activity by detecting the enzymatic products either directly or after amplification of the signal. Other detection assays measure the physical amount of reverse transcriptase by using immunoassays of the enzyme or amplification of the gene encoding the reverse transcriptase.